Abstract
BackgroundMER receptor tyrosine kinase (MERTK) is expressed in a variety of malignancies, including glioblastoma multiforme (GBM). Our previous work demonstrated that inhibition of MERTK using RNA interference induced cell death and chemosensitivity in GBM cells, implicating MERTK as a potential therapeutic target. Here we investigate whether a novel MERTK-selective small molecule tyrosine kinase inhibitor, UNC2025, has similar anti-tumor effects in GBM cell lines.MethodsCorrelations between expression of GAS6, a MERTK ligand, and prognosis were determined using data from the TCGA database. GBM cell lines (A172, SF188, U251) were treated in vitro with increasing doses of UNC2025 (50-400nM). Cell count and viability were determined by trypan blue exclusion. Cell cycle profiles and induction of apoptosis were assessed by flow cytometric analysis after BrdU or Po-Pro-1/propidium iodide staining, respectively. Polyploidy was detected by propidium iodide staining and metaphase spread. Cellular senescence was determined by β-galactosidase staining and senescence-associated secretory cytokine analysis.ResultsDecreased overall survival significantly correlated with high levels of GAS6 expression in GBM, highlighting the importance of TAM kinase signaling in GBM tumorigenesis and/or therapy resistance and providing strong rationale for targeting these pathways in the clinic. All three GBM cell lines exhibited dose dependent reductions in cell number and colony formation (>90% at 200nM) after treatment with UNC2025. Cell cycle analysis demonstrated accumulation of cells in the G2/M phase and development of polyploidy. After extended exposure, 60–80% of cells underwent apoptosis. The majority of surviving cells (65–95%) were senescent and did not recover after drug removal. Thus, UNC2025 mediates anti-tumor activity in GBM by multiple mechanisms.ConclusionsThe findings described here provide further evidence of oncogenic roles for MERTK in GBM, demonstrate the importance of kinase activity for MERTK tumorigenicity and validate UNC2025, a novel MERTK inhibitor, as a potential therapeutic agent for treatment of GBM.
Highlights
Glioblastoma multiforme (GBM) is the most common CNS tumor in adults [1]
Decreased overall survival significantly correlated with high levels of GAS6 expression in GBM, highlighting the importance of TAM kinase signaling in GBM tumorigenesis and/or therapy resistance and providing strong rationale for targeting these pathways in the clinic
The findings described here provide further evidence of oncogenic roles for MER receptor tyrosine kinase (MERTK) in GBM, demonstrate the importance of kinase activity for MERTK tumorigenicity and validate UNC2025, a novel MERTK inhibitor, as a potential therapeutic agent for treatment of GBM
Summary
Glioblastoma multiforme (GBM) is the most common CNS tumor in adults [1]. Patients diagnosed with GBM have a poor prognosis with median survival of ~14 months and a five-year survival rate of less than five percent, even when high dose chemotherapy and radiation are administered. Genomic analyses of resected GBM patient samples are being used to elucidate subgroups and identify abnormal protein and RNA signatures which could serve as novel therapeutic targets for this dismal disease [3, 4]. Several protein targets with the highest expression or most frequent mutations are currently being validated as therapeutic targets and many newly developed or repurposed targeted agents are being evaluated for in preclinical models. If successful, these agents could be moved forward into clinical trials for patients harboring tumors with specific tumor associated or tumor specific antigens, such as EGFR and EGFR variant III, respectively [5]. We investigate whether a novel MERTKselective small molecule tyrosine kinase inhibitor, UNC2025, has similar anti-tumor effects in GBM cell lines
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